This invention relates to the fields of pharmacogenomics, diagnostics and patient therapy. More specifically, the present invention relates to methods of diagnosing and/or treating diseases involving the xcex22-adrenergic receptor or its isoforms.
xcex22-adrenergic receptors (xcex22AR) are G protein coupled receptors that are activated by endogenous catecholamines. When activated by agonists, xcex22AR couple to the G proteins which then signal to effectors such as adenylyl cyclase. These receptors are widely distributed, and play important roles in regulating cardiac, vascular, pulmonary, and metabolic functions. The xcex22AR expressed on cells of the lung act on the bronchial smooth muscle to relax the muscle, thus leading to bronchodilitation. Agonists acting at xcex22AR (clinically referred to as xcex2-agonists) are widely used in the treatment of asthma and chronic obstructive pulmonary diseases (COPD) such as emphysema and chronic bronchitis.
Studies of such physiologic functions of xcex22AR in humans have revealed several observations. First, there appears to be substantial interindividual variation in responsiveness, and secondly receptor function appears to be dynamically regulated as indicated by intraindividual variation. Recently, significant genetic variability in the structure of the xcex22AR in the human population due to single nucleotide polymorphisms (SNPs) in the xcex22AR gene has been delineated (1, 2). These polymorphisms are located at nucleotides 46 (A or G), 79 (C or G), 100 (G or A) and 491 (C or T) of the coding block (where the A of the ATG start codon is designated as nucleotide position 1; GenBank Accession No. AF022956), and result in variation that occurs in the amino-terminus of the receptor at amino acids 16 (Arg or Gly), 27 (Gln or Glu) and 34 (Val or Met) and in the fourth transmembrane spanning domain at amino acid 164 (Thr or Ile). In recombinant cell studies (3, 4), and in primary cultures of cells endogenously expressing these variants (5), clear phenotypic differences have been shown between the polymorphic receptors. The Gly16 receptor was found to undergo enhanced agonist-promoted downregulation of receptor number as compared to the Arg16 receptor (3). In contrast, the Glu27 receptor was found to undergo very little agonist-promoted downregulation compared to the Gln27 receptor (3). These variants are common in the population (1). The Ile64 receptor, which occurs in the heterozygous state in xcx9c5% of the population, displays depressed coupling to the stimulatory G protein, G5 (4).
Subsequent studies have assessed the role of the aforementioned polymorphic xcex22AR in diseases such as asthma [reviewed in (6)], based on the role of xcex22AR in modulating bronchial smooth muscle tone. In these studies, no differences in the frequencies of any of these polymorphisms between non-asthmatics and asthmatics have been reported. However, amino acid variation at positions 16 and 27 were found to act as significant disease modifiers (7-10). In the majority of the above cited studies, the presumption has been that the clinical phenotypes of those with the Gly16 variant were due to enhanced downregulation of this receptor (as compared to those with the Arg16 receptor) by endogenous catecholamines. Thus responsiveness in individuals with this polymorphism has been considered depressed due to this tonic downregulation. A similar scenario is considered in those with the Glu27 variant, where responsiveness is greater than those with the Gln27 receptor, presumably due to its minimal downregulation by catecholamines. An amplification of these differences may occur during chronic agonist administration, as has recently been shown in asthma (11).
Previous studies (4) with the agonists epinephrine, norepinephrine, and isoproterenol have shown that the Ile164 receptor displays a small decrease in binding affinity to the receptor as determined in competition binding studies with [125I]cyanopindolol ([125I]cyp) in the presence of GTP. Similarly, binding curves with isoproterenol in the absence of GTP were different between the two receptors (FIG. 2). This indicates that binding of these agents at the classic agonist binding domains of the xcex22AR in transmembrane segments 3 and 5 of the receptor is of lower affinity. However, they do not provide information on the binding affinities of other agonists, which have diverse structures and are commonly used to treat lung disease, or the duration of action of such agonists or interactions of such agonists to other parts of the receptor. The nature of the Ile164 receptor is further clouded by an examination of the ability of the Ile164 receptor to stimulate adenylyl cyclase compared to the more prevalent (Thr164) receptor. For example, studies show that the agonist epinephrine produces decreased stimulation while the agonist dopamine displays normal binding affinities and does not show decreased stimulation. Therefore, the effects of substituting Ile for Thr at amino acid 164 on biological activity of the xcex22AR receptor in vitro or in vivo are unclear.
Salmeterol, a unique agonist used in the treatment of asthma and COPD, has been shown to interact with the human xcex22AR (12). Salmeterol has a long duration of action (xcx9c12 hrs) after a single administration. The long side chain of the molecule (see FIG. 3) interacts with the receptor in a manner that tethers the molecule to the fourth transmembrane spanning domain. This region is termed the xe2x80x9cexositexe2x80x9d because it is outside of the agonist binding sites of traditional agonists. Once anchored, the amine and catechol groups of the molecule, repetitively, interact with the active binding site in transmembrane domains 3 and 5. This was determined by making chimeric xcex22-xcex21AR mutants which allowed definition of the general area where salmeterol interacts in this unique way with the xcex22AR. Studies of the interaction between salmeterol and chimeric xcex22ARs having altered exosite regions indicate that salmeterol derives its increased duration of action from an interaction with the exosite region. However, in these studies, threonine was the amino acid at position 164.
Because xcex2-agonists are the most commonly prescribed therapeutic for asthma and pharmaceutical companies continue to introduce new xcex2-agonists to the market, it would be useful to determine the effect of polymorphisms in the human xcex22AR gene on receptor response to these drugs.
The present invention is based on the surprising discoveries (1) that the xcex2-agonists salmeterol, albuterol, metaproterenol, terbutaline and formoterol activate the Thr164 variant of xcex22AR to a higher level than the Ile164 variant and (2) that the Ile 164 variant exhibits reduced exosite binding and shorter duration of action to salmeterol. It is believed that individuals expressing the Ile164 variant will exhibit reduced response to these xcex2-agonists and, in the case of salmeterol, will also exhibit a reduced duration of response. As all these xcex2-agonists are currently used in the treatment of patients suffering from diseases modified by the xcex22AR, particularly asthma and COPD, knowledge of a patient""s genotype for the polymorphic site at nucleotide +491 (+491PS) in the xcex22AR gene would be useful in prescribing appropriate xcex2-agonist therapy.
Thus, the present invention provides diagnostic methods for predicting a patient""s bronchodilating response to an agonist of xcex22AR. In one embodiment, the method comprises determining the patient""s genotype for the +491PS, wherein a patient who has at least one thymine at this site is likely to exhibit a poor response to the agonist. In another embodiment, the method comprises assaying a sample from the patient for expression of the Ile164 xcex22AR variant, wherein presence of the Ile164 xcex22AR variant indicates the patient is likely to respond poorly to the agonist. In preferred embodiments of the diagnostic method, the agonist is salmeterol, albuterol, metaproterenol, terbutaline or formoterol.